[Alexander James Phillips]

Sorry I realised I forgot to give this feedback:

While GITC + detergent buffers are expected to inactivate coronaviruses (e.g. MERS-CoV, Kumar et al., 2015 https://www.ncbi.nlm.nih.gov/pubmed/26190637 ), this has not been definitively tested for SARS-CoV-2.

Note that Kumar et al. paper actually uses GITC (guanidinium isothiocyanate) containing AVL __with ethanol__:

AVL buffer (Qiagen) at a 4:1 ratio (400 μl AVL:100 μl supernatant) and incubated at room temperature for 10 min. Ethanol (>95%) was added to a final volume of 900 μl and vortexed.

So I believe it was misleading of Kumar et al. to say AVL inactivated the MERS virus.

Cheers,

AJP

p.s. there is more conversation here (as well as in other places) on lysis buffer inactivation of SARS-CoV-2: https://testingmethods.crowdicity.com/post/3161470

[Phil Oberacker]

@Alexander James Phillips, do these aggregates look a bit like flakes that form right after the addition of alcohol? I encountered this while testing bead capacity and uploaded the respective results on researchgate (https://www.researchgate.net/project/Bio-On-Magnetic-Beads-BOMB). Just check the update at the 26th of February. Simple reason would be overloaded beads.

Cheers
Phil

[OMC Healthcare]

I am wondering what type of Antifoam I should use in GITC Buffer preparation. There are different types of antifoam reagent & I am confused which one will be appropriate for this application.

[Phil Oberacker]

As specified in #8.2 e.g., we use Antifoam 204 from Merck (PN: A8311-50ml).

[OMC Healthcare]

We have ordered Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophylic), 100 mL (Cat#45152105050350) from Cytiva ( https://www.cytivalifesciences.com/en/us). And the costomer service spoke with me regarding the purpose of the bead to use & the gave following suggestion-

———————————-

One point, This is not a popular bead. This one does have some sign of PCR inhibitor for some work, so is not one we would recommend. Especially for COVID19, we have no demand for this at all. Wondering why you pick this?

If you want better speed (speedbead double layer magnetic) than the regular bead (single layer), then may consider 6515 bead (so 100ml would be Cat#6515210505350).

————————————

Please advise.

 

 

[OMC Healthcare]

Can you provide information about the chemical you are using with manufacturer & cat# (paramagnetic beads, GITC, Sarkosyl, EDTA etc.). I really want to get the similar result what you are getting with this BOMB protocol.

[TimH]

Hi OMC Healthcare – this bead works very well in our hands, but other beads are also likely to work well (including self-fabricated beads, as in our protocols). We’ve not detected any sign of PCR inhibitor – remember that the beads are washed in the preparation steps, and then go through purification involving more washes, before being resuspended in MQH20. So I’m not sure where this inhibition would be coming from but happy to hear more details if the customer service rep will provide it.

Best wishes,

TimH

[TimH]

You can find this information in protocol #6.1 on our main page, or find below in the poorly formatted copy/paste from this page. But chemicals of good quality sourced elsewhere will very likely be fine. (note, you have details of the beads already)

Ethanol (C2H6O, 99.9%) Honeywell/ Riedel-de Haёn 34963

Guanidine isothiocyanate (GITC, C2H6N4S) Roth Chemicals 2628.4

Tris(hydroxymethyl)- aminomethane (Tris, C4H11NO3) Roth Chemicals AE15.3

N-Lauroylsarcosine sodium salt (Sarkosyl, C15H29NO3Na) Sigma (Merck) L9150- 50G 2

Antifoam 204 Sigma (Merck) A8311- 50ML

Ethylenediamine tetraacetic acid disodium salt dihydrate (EDTA (C10H14N2Na2O8 · 2 H2O) Roth Chemicals

Isopropanol (C3H8O) Acros Organic 18413002 5

[Peter Stoilov]

Hi all,

 

first of all thanks to the BOMB team for the excellent protocol!

We tested a slightly modified protocol for RNA extraction efficiency and sensitivity using in vitro transcribed RNA for the SARS-nCov2 virus N-gene (CDC primer sets).

The modifications are as followed:

1. GITC solution is diluted by 10% to prevent precipitation. The volume is adjusted accordingly to 110ul lysis buffer per 200ul of sample.

2. The magnetic beads are diluted 25 fold, and their volume is reduced by two fold. This compensates for the increase of the GITC solution volume, so that the volumes for rest of the reagents do not need to be changed.

3. Incubation time on the magnet is longer: up to 25-30minutes with mixing at 10min. This depends on the strength of the magnet, so you have to pay attention and make sure you pulled down all of the beads before you start washing. I suspect this is the reason why some see lower recovery than column kits.

4. We use water instead of TE to elute the nucleic acids. We attempted to reduce the elution volume, but at least on our plates it was hard to recover the liquid when the volume was too low so we stick with 50ul elution volume.

5. We multiplexed the CDC primer set and put all three primer sets together with FAM, VIC and ABY labeled TaqMan probes.

6. We use Thermo TaqPath single tube mastermix (4x) and put 10ul of the eluted RNA per reaction.

 

With the protocol modified as described we see near complete recovery of the RNA. The limit of detection is 145 copies per ml (call it 200 if you want to be conservative). We call a positive hit if at least one out of the three probes gives us a positive signal. At 145 copies per ml 90% of the reaction are positive for at least 2 probes and 100% are positive for at least one probe. At 15 copies per ml 50% of the reactions are positive for at least one probe. To put this in perspective, at 15 copies/ml starting material before the purification and assuming 100% recovery, a PCR reaction would contain 0.6 copies of RNA meaning it has 60% chance to have one RNA molecule. The calculation is as follows: 200ul x 15 copies/ml= 3 copies; 10ul out of  50ul containing 3 copies = 0.6 copies per reaction.

We used a simulated virus, lentiviral particles with cloned SARS-nCov2-N gene in VTM, and the viral RNA also seems to be recovered efficiently. I will have hard data on the LOD for the viral particles in the near future.

The sensitivity can be increased by using larger volumes. Deep well plates with capacity of 2ml will in theory allow using 400-500ul of sample and double the sensitivity. The downside is that this  setup is harder to handle: deep well plates require longer tips, make it harder to reach the bottom, don’t sit well on the magnets, and the volumes exceed the capacity of the multichannel pipettes thus requiring more pipetting.

Finally, we did tests on the Qiagen viral RNA extraction kits and the recovery there is 67% of the input. So, in our hands the BOMB-COVID19 protocol is performing better that the Qiagen kit with the added benefit of not needing carrier RNA (does anyone know the origin of the carrier RNA in the Qiagen kits?).

 

[Alexander James Phillips]

== Technology adoption – who’s using what variant, where, for what ==

Hi all,

I went through the different forum posts to see who has said what and tried to guess where you’re all at with the Bomb.Bio protocol for RNA purification. Specifically I’m looking for technicians, scientists, or managers at public pathology labs, academic researchers, or private companies who are:

1. Evaluating (the Bomb.bio RNA purification technique for clinical sample testing with or without automation)
2. Deployment in progress (for clinical sample testing)
3. Clinical sample testing

Please correct / add your details here: https://docs.google.com/spreadsheets/d/13_cSMSHtzMu0l1TCJF3KzCdXo88ZPCc1OqSKuAnQAQQ/edit#gid=0

It’s purpose is to:

1. allow other pathology lab staff to get a sense for the adoption of the technique
2. to make them feel more confident they’re going to be able to do something worthwhile
3. and that there’s lots of other people / labs out there who can support them if they have problems implementing it. (I know the Bomb.bio team are very responsive on the forum anyway but they don’t necessarily know that).

Thank you!
AJP

[Sheila Ons]

Hello, thanks for providing this protocol. If I use silica-coated beads, is there some step on this protocol that I must modify, or take extra-care? Or is exactly the same protocol for both kind of beads? Thank you in advance for your answer.

 

Sheila Ons

[Phil Oberacker]

Hi Sheila,

The COVID-19 protocol and the used buffers are based on our RNA isolation protocol #8.2, in which we purify RNA with silica-beads, so there shouldn’t be a problem. However, to my knowledge, we didn’t try this exact protocol with silica-beads yet, so no guarantee. But I don’t see why it wouldn’t work. Just give it a try 🙂 if you have any more questions, don’t hesitate to ask.

Cheers,
Phil

[Sheila Ons]

Thanks Phil! I will let you know how it works once we tried.

Cheers.

 

Sheila

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